ABSTRACT Background: The mammalian heart beats spontaneously and autonomously due to few thousand (~10,000) pacemaker cells. Although we have a general understanding of how individual cardiac pacemaker cells beat automatically, there is a lack of understanding in how a few pacemaker cells can drive the beating of the entire heart. This problem, known as a ?source-sink mismatch?, is a fundamental concept that has been difficult to study due to it being painfully low-throughput to study these pacemaker cells. This is because no testable model of the SAN exists, incorporating the cardiac pacemaker cells and quiescent cardiomyocytes. Typically, just a handful of native pacemaker cells can be isolated from the native SAN, and the isolated cell cannot be cultured. Recently, my group has demonstrated conversion of ordinary ventricular cardiomyocytes to induced pacemaker cells (iPCs) by singular expression of TBX18. In this proposal, we seek to engineer tissue models of the SAN by exploiting the de novo iPCs. We hypothesize that 2- and 3-dimensional architectures of the iPCs may serve as in vitro models of native SANs. Approach: We will examine four design principles of the native SAN, i) minimum number of iPCs required to pace a given number of neighboring ventricular myocytes, ii) role of non-myocyte population in pacemaking, iii) shape of the SAN, and iv) the need for exit pathways. Our 2D model will consist of patterned monolayers while our 3D model uses patterned cardiac spheroids. Using routine polydimethylsiloxane (PDMS) stenciling techniques, we will create a population of iPCs enclosed by a population of quiescent ventricular cardiomyocytes. The major readouts are i) real-time, whole-cell Ca2+ transients of the entire monolayers, ii) fast, high-resolution optical mapping of the monolayers with a voltage-sensitive dye, and iii) macro-scale, multi-electrode array measurements of field potentials. Our preliminary data indicate that iPC-spheroids are viable for at least three weeks. When a cluster of 15-20 TBX18 spheroids was surrounded by a monolayer of ventricular myocytes, TBX18, but not GFP (control), spheroids were able to pace and drive the neighboring sheet of ventricular myocytes. Successful completion of our project can lead to creation of engineered SA nodes (eSANs) that recapitulate the design principles of the native SAN. In turn, this technology provides a convenient platform on which other SAN design principles may be built toward persistent biological pacemakers.